Predicting Experimental B22 Values and the Effects of Histidine Charge States for Monoclonal Antibodies Using Coarse-Grained Molecular Simulations.

Static light scattering (SLS) was used to characterize five monoclonal antibodies (MAbs) as a function of total ionic strength (TIS) at pH values between 5.5 and 7.0. Second osmotic virial coefficient (B22) values were determined experimentally for each MAb as a function of TIS using low protein concentration SLS data. Coarse-grained molecular simulations were performed to predict the B22 values for each MAb at a given pH and TIS. To include the effect of charge fluctuations of titratable residues in the B22 calculations, a statistical approach was introduced in the Monte Carlo algorithm based on the protonation probability based on a given pH value and the Henderson-Hasselbalch equation. The charged residues were allowed to fluctuate individually, based on the sampled microstates and the influence of electrostatic interactions on net protein-protein interactions during the simulations. Compared to static charge simulations, the new approach provided improved results compared to experimental B22 values at pH conditions near the pKa of titratable residues.

Reduction Rate of 1-Phenyl Phospholane 1-Oxide Enhanced by Silanol Byproducts: Comprehensive DFT Study and Kinetic Modeling Linked to Reagent Design.

Important stoichiometric transformations like Wittig and Appel reactions have been implemented in a catalytic fashion in the past decade. The phosphine oxide generated in situ can be reintroduced as phosphine into the catalytic cycle using mild and selective silane reagents (redox-driven catalysis). While the field of experimental investigation has been fully expanding in the past decade, theoretical studies are still sparse. In this present work, density functional theory (DFT) has been used to characterize the free energy surfaces of the reduction of 1-phenyl phospholane 1-oxide with four different silanes. Found stationary points have been studied in-depth to highlight mechanistic peculiarities, like the effect of substituents at the silicon center and the parallel and competitive reactivity between the precursor silanes and their semioxidized byproducts. Calculated thermodynamic parameters in combination with "real" values for concentrations have been used in the formulation of rate equations for simple bimolecular and monomolecular steps of the mechanism. The deterministic integration concentrations versus time of such rate equations led to a realistic description of the systems under study and paved the way to strategic and rational design of new silanes with increased reactivity.

Catalytic wittig reactions of semi- and nonstabilized ylides enabled by ylide tuning.

The first examples of catalytic Wittig reactions with semistabilized and nonstabilized ylides are reported. These reactions were enabled by utilization of a masked base, sodium tert-butyl carbonate, and/or ylide tuning. The acidity of the ylide-forming proton was tuned by varying the electron density at the phosphorus center in the precatalyst, thus facilitating the use of relatively mild bases. Steric modification of the precatalyst structure resulted in significant enhancement of E selectivity up to >95:5, E/Z.

Breaking the ring through a room temperature catalytic Wittig reaction.

One ring no longer rules them all: Employment of 2.5-10 mol % of 4-nitrobenzoic acid with phenylsilane led to the development of a room temperature catalytic Wittig reaction (see scheme). Moreover, these enhanced reduction conditions also facilitated the use of acyclic phosphine oxides as catalysts for the first time. A series of alkenes were produced in moderate to high yield and selectivity.

Part I: the development of the catalytic Wittig reaction.

We have developed the first catalytic (in phosphane) Wittig reaction (CWR). The utilization of an organosilane was pivotal for success as it allowed for the chemoselective reduction of a phosphane oxide. Protocol optimization evaluated the phosphane oxide precatalyst structure, loading, organosilane, temperature, solvent, and base. These studies demonstrated that to maintain viable catalytic performance it was necessary to employ cyclic phosphane oxide precatalysts of type 1. Initial substrate studies utilized sodium carbonate as a base, and further experimentation identified N,N-diisopropylethylamine (DIPEA) as a soluble alternative. The use of DIPEA improved the ease of use, broadened the substrate scope, and decreased the precatalyst loading. The optimized protocols were compatible with alkyl, aryl, and heterocyclic (furyl, indolyl, pyridyl, pyrrolyl, and thienyl) aldehydes to produce both di- and trisubstituted olefins in moderate-to-high yields (60-96%) by using a precatalyst loading of 4-10 mol%. Kinetic E/Z selectivity was generally 66:34; complete E selectivity for disubstituted α,ß-unsaturated products was achieved through a phosphane-mediated isomerization event. The CWR was applied to the synthesis of 54, a known precursor to the anti-Alzheimer drug donepezil hydrochloride, on a multigram scale (12.2 g, 74% yield). In addition, to our knowledge, the described CWR is the only transition-/heavy-metal-free catalytic olefination process, excluding proton-catalyzed elimination reactions.

Use of cyclin D1 in conjunction with human papillomavirus status to predict outcome in oropharyngeal cancer.

There is increasing use of multiple molecular markers to predict prognosis in human cancer. Our aim was to examine the prognostic significance of cyclin D1 and retinoblastoma (pRb) expression in association with human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma. Clinical records and specimens of 226 patients with follow-up from 1 to 235 months postdiagnosis were retrieved. Tumor HPV status was determined by HPV E6-targeted multiplex real-time PCR/p16 semiquantitative immunohistochemistry and cyclin D1 and pRb expression by semiquantitative immunohistochemistry. Determinants of recurrence and mortality hazards were modeled using Cox regression with censoring at dates of last follow-up. The HPV-positivity rate was 37% (91% type 16). HPV was a predictor of recurrence, an event (recurrence or death) and death after adjustment for clinicopathological variables. There were inverse relationships between HPV status and cyclin D1 and pRb. On univariate analysis, cyclin D1 predicted locoregional recurrence, event and death and pRb predicted event and death. Within the HPV-positive group, after adjusting for clinicopathological factors, patients with cyclin D1-positive cancers had up to a eightfold increased risk of poor outcome relative to those with cyclin D1-negative tumors. However, within the HPV-negative group, there was only a very small adjusted increased risk. A combination of pRb and HPV did not provide additional prognostic information. Our data provide the first evidence that a combination of HPV and cyclin D1 provides more prognostic information in oropharyngeal cancer than HPV alone. If findings are confirmed, treatment based on HPV and cyclin D1 may improve outcomes.

Structure-activity relationship analysis of Pd-PEPPSI complexes in cross-couplings: a close inspection of the catalytic cycle and the precatalyst activation model.

A series of Pd-N-heterocyclic carbene (Pd-NHC) complexes with various NHC, halide and pyridine ligands (PEPPSI (pyridine, enhanced, precatalyst, preparation, stabilisation and initiation) precatalysts) were prepared, and the effects of these ligands on catalyst activation and performance were studied in the Kumada-Tamao-Corriu (KTC), Negishi, and Suzuki-Miyaura cross-coupling reactions. The lowered reactivity of more hindered 2,6-dimethylpyridyl complex 4 in the Negishi and KTC reactions is consistent with slow reductive dimerisation of the organometallic reaction partner during precatalyst activation. Comparative rate studies of complexes 1, 4 and 5 in the KTC and Suzuki-Miyaura reactions verify that 4 activated more slowly than the others. A potential on/off mechanism of pyridine coordination to NHC-Pd(0) is also plausible, in which the more basic pyridine stays bound for longer.

Why do silanes reduce electron-rich phosphine oxides faster than electron-poor phosphine oxides?

Organophosphine-mediated reactions that generate P[double bond, length as m-dash]O-bonded byproducts can be transformed into catalytic processes by reducing the R3P[double bond, length as m-dash]O byproduct back to PR3in situ with a silane. DFT calculations explain why the most readily reduced phosphine oxides are those incorporating electron-rich (e.g. alkyl) substituents rather than electron-deficient (e.g. aryl) substituents.

Physiological effects of high-flow nasal cannula therapy in preterm infants.

OBJECTIVE: High-flow nasal cannula (HFNC) therapy is increasingly used in preterm infants despite a paucity of physiological studies. We aimed to investigate the effects of HFNC on respiratory physiology. STUDY DESIGN: A prospective randomised crossover study was performed enrolling clinically stable preterm infants receiving either HFNC or nasal continuous positive airway pressure (nCPAP). Infants in three current weight groups were studied: <1000 g, 1000-1500 g and >1500 g. Infants were randomised to either first receive HFNC flows 8-2 L/min and then nCPAP 6 cm H2O or nCPAP first and then HFNC flows 8-2 L/min. Nasopharyngeal end-expiratory airway pressure (pEEP), tidal volume, dead space washout by nasopharyngeal end-expiratory CO2 (pEECO2), oxygen saturation and vital signs were measured. RESULTS: A total of 44 preterm infants, birth weights 500-1900 g, were studied. Increasing flows from 2 to 8 L/min significantly increased pEEP (mean 2.3-6.1 cm H2O) and reduced pEECO2 (mean 2.3%-0.9%). Tidal volume and transcutaneous CO2 were unchanged. Significant differences were seen between pEEP generated in open and closed mouth states across all HFNC flows (difference 0.6-2.3 cm H2O). Infants weighing <1000 g received higher pEEP at the same HFNC flow than infants weighing >1000 g. Variability of pEEP generated at HFNC flows of 6-8 L/min was greater than nCPAP (2.4-13.5 vs 3.5-9.9 cm H2O). CONCLUSIONS: HFNC therapy produces clinically significant pEEP with large variability at higher flow rates. Highest pressures were observed in infants weighing <1000 g. Flow, weight and mouth position are all important determinants of pressures generated. Reductions in pEECO2 support HFNC's role in dead space washout.

Prognostic significance of vascular endothelial growth factor in squamous cell carcinomas of the tonsil in relation to human papillomavirus status and epidermal growth factor receptor.

BACKGROUND: Angiogenesis markers, vascular endothelial growth factor (VEGF) and microvessel density (MVD) have been associated with prognosis in squamous cell carcinomas (SCCs) of the head and neck. Other prognostic variables such as human papillomavirus (HPV) and epidermal growth factor (EGFR) may also be involved in tumour angiogenesis. This study determined relationships between VEGF, MVD, EGFR, HPV, response to radiotherapy and clinical outcome in 85 tonsillar SCCs. METHODS: HPV status was determined by an HPV multiplex real-time polymerase chain reaction (PCR) assay/p16 immunohistochemistry. Expression of VEGF, CD31 (as marker of MVD) and EGFR was assessed by semiquantitative immunohistochemistry. RESULTS: Strong VEGF expressers were significantly more likely to have higher MVD than were weak expressers. There were no associations between VEGF or MVD and gender, patient age, TNM stage, EGFR expression or HPV status. Tumours with MVD of >15 per high-power field were significantly more likely to be poorly differentiated. There was a significant inverse relationship between EGFR and HPV status. HPV was a strong independent marker of loco-regional recurrence and death. VEGF and EGFR were risk factors for local recurrence and disease-specific death on univariate analysis but the associations weakened after adjustment for HPV. Among patients treated with radiotherapy, VEGF was associated with disease-specific death after adjusting for HPV and TMN stage. High-VEGF-expressing tumours positive for EGFR had a worse prognosis than all other groups combined after adjusting for HPV and TNM stage. CONCLUSIONS: HPV is a stronger prognostic marker than VEGF or EGFR in tonsillar SCCs. VEGF correlates with MVD in these tumours.

Density functional theory investigation of the alkyl-alkyl Negishi cross-coupling reaction catalyzed by N-heterocyclic carbene (NHC)-Pd complexes.

A novel mechanism is proposed for the Pd-1,3-(2,6-diisopropylphenyl)imidazolyl-2-ylidene (1) catalyzed Negishi reaction. DFT computations supported by atoms-in-molecules (AIM) analyses of non-truncated models show that a "steric wall" created by the N-substituent on the ligand guides reactants to and from the Pd center. Notably, transmetalation and not oxidative addition is found to be rate-limiting. Additionally, a key Pd-Zn interaction (approximately = 2.4 A, rho(b) approximately = 0.0600 au) is identified in the mechanism. This interaction persists beyond reductive elimination and, in combination with the ligand, facilitates reductive elimination by creating a highly sterically crowded environment in the coordination sphere of the Pd center.

Adjuvant postoperative radiotherapy to the cervical lymph nodes in cutaneous melanoma: is there any benefit for high-risk patients?

BACKGROUND: The use of adjuvant radiotherapy after lymph node dissection for metastatic melanoma remains controversial. This study examined the effectiveness of adjuvant radiotherapy in controlling regional disease in high-risk patients. METHODS: A total of 716 patients were identified from a large prospective database who underwent cervical lymph node surgery between 1990 and 2004. Patients with high-risk disease were offered radiotherapy (n = 129), and this group was compared with the group of patients who did not receive radiotherapy (n = 587) in the same period. RESULTS: Radiotherapy did not improve regional control in patients who had metastatic melanoma of the cervical lymph nodes (P = .2). There were 10% fewer regional recurrences in patients with extracapsular spread who received adjuvant radiotherapy, although this was not statistically significant (P = .34). Adjuvant radiotherapy conferred no overall survival benefit to patients with nodal metastases (P = .39). There was a statistically significant trend for worse survival with increasing nodal tumor burden that remained unchanged with adjuvant radiotherapy. CONCLUSION: This large, nonrandomized retrospective study found no evidence to support the use of adjuvant radiotherapy for high-risk melanoma. A multicenter randomized, controlled trial investigating this important clinical dilemma is advocated.

High yielding alkylations of unactivated sp3 and sp2 centres with alkyl-9-BBN reagents using an NHC-based catalyst: Pd-PEPPSI-IPrf.

High yielding, room temperature cross couplings of unactivated alkyl bromides and aryl bromides/chlorides with alkyl-9-BBN reagents has been achieved using an NHC-based catalyst (Pd-PEPPSI-IPr) via a general, functional-group tolerant and easily implemented protocol.

The prognostic impact of extent of bone invasion and extent of bone resection in oral carcinoma.

OBJECTIVE: The prognostic impact of mandibular invasion by oral squamous cell carcinoma (SCC) is controversial. The objective of this study was to assess the effect that extent of bone invasion has on recurrence and survival in patients treated with marginal and segmental mandible resection. STUDY DESIGN: A retrospective cohort study of patients whose clinicopathologic information had been collected prospectively into a dedicated head and neck database. METHODS: Local control and disease-specific survival were retrospectively reviewed in 111 patients (median follow-up, 44 months) with oral SCC undergoing marginal or segmental mandibulectomy. RESULTS: Bone invasion was present in 46% of marginal and 94% of segmental resections. Five-year local control was similar following marginal (83%) and segmental mandibulectomy (86%). There was no correlation with presence or extent of bone invasion. Survival at 5 years was 71% and this correlated with bone invasion and involved margins (P < .05), but not with extent of mandible invasion or resection. CONCLUSIONS: Marginal mandibulectomy does not adversely alter outcome in selected patients with bone invasion provided margins are not compromised.

Biophysical characterization and molecular simulation of electrostatically driven self-association of a single-chain antibody.

Colloidal protein-protein interactions (PPI) are often expected to impact key behaviors of proteins in solution, such as aggregation rates and mechanisms, aggregate structure, protein solubility, and solution viscosity. PPI of an anti-fluorescein single chain antibody variable fragment (scFv) were characterized experimentally at low to intermediate ionic strength using a combination of static light scattering and sedimentation equilibrium ultracentrifugation. Surprisingly, the results indicated that interactions were strongly net-attractive and electrostatics promoted self-association. Only repulsive interactions were expected based on prior work and calculations based a homology model of a related scFv crystal structure. However, the crystal structure lacks the charged, net-neutral linker sequence. PyRosetta was used to generate a set of scFv structures with different linker conformations, and coarse-grained Monte Carlo simulations were used to evaluate the effect of different linker configurations via second osmotic virial coefficient (B22 ) simulations. The results show that the configuration of the linker has a significant effect on the calculated B22 values, and can result in strong electrostatic attractions between oppositely charged residues on the protein surface. This is particularly relevant for development of non-natural antibody products, where charged linkers and other loop regions may be prevalent. The results also provide a preliminary computational framework to evaluate the effect of unstructured linkers on experimental protein-protein interaction parameters such as B22 .

New nanoscale toughening mechanisms mitigate embrittlement in binary nanocrystalline alloys.

Nanocrystalline metals offer significant improvements in structural performance over conventional alloys. However, their performance is limited by grain boundary instability and limited ductility. Solute segregation has been proposed as a stabilization mechanism, however the solute atoms can embrittle grain boundaries and further degrade the toughness. In the present study, we confirm the embrittling effect of solute segregation in Pt-Au alloys. However, more importantly, we show that inhomogeneous chemical segregation to the grain boundary can lead to a new toughening mechanism termed compositional crack arrest. Energy dissipation is facilitated by the formation of nanocrack networks formed when cracks arrested at regions of the grain boundaries that were starved in the embrittling element. This mechanism, in concert with triple junction crack arrest, provides pathways to optimize both thermal stability and energy dissipation. A combination of in situ tensile deformation experiments and molecular dynamics simulations elucidate both the embrittling and toughening processes that can occur as a function of solute content.

Palladium complexes of N-heterocyclic carbenes as catalysts for cross-coupling reactions--a synthetic chemist's perspective.

Palladium-catalyzed C-C and C-N bond-forming reactions are among the most versatile and powerful synthetic methods. For the last 15 years, N-heterocyclic carbenes (NHCs) have enjoyed increasing popularity as ligands in Pd-mediated cross-coupling and related transformations because of their superior performance compared to the more traditional tertiary phosphanes. The strong sigma-electron-donating ability of NHCs renders oxidative insertion even in challenging substrates facile, while their steric bulk and particular topology is responsible for fast reductive elimination. The strong Pd-NHC bonds contribute to the high stability of the active species, even at low ligand/Pd ratios and high temperatures. With a number of commercially available, stable, user-friendly, and powerful NHC-Pd precatalysts, the goal of a universal cross-coupling catalyst is within reach. This Review discusses the basics of Pd-NHC chemistry to understand the peculiarities of these catalysts and then gives a critical discussion on their application in C-C and C-N cross-coupling as well as carbopalladation reactions.

Cystic fibrosis presenting with bilateral facial palsy.

A 15-week old male infant presented with bilateral lower motor neuron facial palsy of unknown cause. Subsequently his growth deteriorated and he developed progressively worsening cough and wheeze. A diagnosis of cystic fibrosis was confirmed and hypovitaminosis A detected. Improvement of the facial palsy was noted following standard management of cystic fibrosis including vitamin A supplementation.

New evidence for geographic variation in the role of human papillomavirus in tonsillar carcinogenesis.

AIMS: Our previous studies of tonsillar cancers from New South Wales, Australia, and Jilin Province in the north-east of China, provided evidence that the proportion of these cancers attributable to human papillomavirus (HPV) varies geographically. This study provides the first data on HPV in tonsillar cancers from Hong Kong. METHODS: A total of 49 Hong Kong tonsillar cancers were analysed for HPV DNA by PCR/sequencing and for p16(INK4A), retinoblastoma (pRb) protein, cyclin D1 and p53 expression by semiquantitative immunohistochemistry as evidence of virus causality. Results were compared with those from New South Wales and Jilin Province. RESULTS: Of the 31 Hong Kong cancers with amplifiable DNA, nine (29%) were HPV positive by PCR compared with 46% from New South Wales and 0% from Jilin Province. HPV positivity correlated with female gender, young age, over-expression of p16(INK4A) and loss of pRb and cyclin D1. Five-year disease-specific survival for patients with HPV positive and HPV negative cancers was 82 and 42%, respectively. Relationships between HPV status and cell protein expression in Hong Kong cancers were consistent with those from New South Wales and Jilin Province. The proportion of HPV-associated cancers reflected the relative incidence of oropharyngeal cancer in these regions. CONCLUSIONS: HPV is responsible for a small proportion of tonsillar cancers in Hong Kong patients. Differences in the proportions of tumours attributable to HPV in Hong Kong, New South Wales and Jilin Province may be due to environmental, cultural or genetic factors in the different populations.

Clinical outcome after surgery for 75 patients with parotid sialadenitis.

OBJECTIVE: To analyze the clinical outcome of patients treated surgically for chronic parotid sialadenitis. STUDY DESIGN: A retrospective cohort study of patients whose clinicopathologic information had been collected prospectively onto a dedicated head and neck database. METHODS: Between 1987 and 2006, a total of 75 patients had 78 parotidectomies, 17 superficial and 61 near-total, to treat chronic parotid sialadenitis. The clinicopathologic data including treatment morbidity and outcome were analyzed. RESULTS: Temporary postoperative facial weakness occurred in 26 (33%) patients, and this was permanent, although partial, in one (1%) patient. There was no significant difference (P > .05) in the incidence of temporary facial nerve neuropraxia in the superficial (35%) and near-total parotidectomy (33%) groups. Recurrence of symptoms was noted in two patients who had undergone near-total parotidectomy and none of the patients who had undergone superficial parotidectomy. Chronic parotid sialadenitis was effectively treated in 97% of patients undergoing parotidectomy. CONCLUSION: Near-total parotidectomy is a safe and efficacious surgical treatment in the management of patients with debilitating severe chronic parotid sialadenitis.